The Complement System
The complement system is a complex enzyme cascade made up of a series of serum glycoproteins that normally exist in inactive, pro-enzyme form. Three main pathways, the classical, alternative and mannose-binding lectin pathway, can activate complement, which merge at the level of where two similar C3 convertases cleave C3 into C3a and C3b.
Macrophages are specialist cells that have developed an innate capacity to recognize subtle differences in the structure of cell-surface expressed identification tags, so called molecular patterns (Taylor, et al., Eur J Immunol 33, 2090-1097 (2003); Taylor, et al., Annu Rev Immunol 23, 901-944 (2005)). While the direct recognition of these surface structures is a fundamental aspect of innate immunity, opsonization allows generic macrophage receptors to mediate engulfment, increasing the efficiency and diversifying recognition repertoire of the phagocyte (Stuart and Ezekowitz, Immunity 22, 539-550 (2005)). The process of phagocytosis involves multiple ligand-receptor interactions, and it is now clear that various opsonins, including immunoglobulins, collectins, and complement components, guide the cellular activities required for pathogen internalization through interaction with macrophage cell surface receptors (reviewed by Aderem and Underhill, Annu Rev Immunol 17, 593-623 (1999); Underhill and Ozinsky, Annu Rev Immunol 20, 825-852 (2002)). While natural immunoglobulins encoded by germline genes can recognize a wide variety of pathogens, the majority of opsonizing IgG is generated through adaptive immunity, and therefore efficient clearance through Fc receptors is not immediate (Carroll, Nat Immunol 5, 981-986 (2004)). Complement, on the other hand, rapidly recognizes pathogen surface molecules and primes the particle for uptake by complement receptors (Brown, Infect Agents Dis 1, 63-70 (1991)).
Complement consists of over 30 serum proteins that opsonize a wide variety of pathogens for recognition by complement receptors. Depending on the initial trigger of the cascade, three pathways can be distinguished (reviewed by (Walport, N Engl J Med 344, 1058-1066 (2001)). All three share the common step of activating the central component C3, but they differ according to the nature of recognition and the initial biochemical steps leading to C3 activation. The classical pathway is activated by antibodies bound to the pathogen surface, which in turn bind the C1q complement component, setting off a serine protease cascade that ultimately cleaves C3 to its active form, C3b. The lectin pathway is activated after recognition of carbohydrate motifs by lectin proteins. To date, three members of this pathway have been identified: the mannose-binding lectins (MBL), the SIGN-R1 family of lectins and the ficolins (Pyz et al., Ann Med 38, 242-251 (2006)) Both MBL and ficolins are associated with serine proteases, which act like C1 in the classical pathway, activating components C2 and C4 leading to the central C3 step. The alternative pathway contrasts with both the classical and lectin pathways in that it is activated due to direct reaction of the internal C3 ester with recognition motifs on the pathogen surface. Initial C3 binding to an activating surface leads to rapid amplification of C3b deposition through the action of the alternative pathway proteases Factor B and Factor D. Importantly, C3b deposited by either the classical or the lectin pathway also can lead to amplification of C3b deposition through the actions of Factors B and D. In all three pathways of complement activation, the pivotal step in opsonization is conversion of the component C3 to C3b. Cleavage of C3 by enzymes of the complement cascades exposes the thioester to nucleophilic attack, allowing covalent attachment of C3b onto antigen surfaces via the thioester domain. This is the initial step in complement opsonization. Subsequent proteolysis of the bound C3b produces iC3b, C3c and C3dg, fragments that are recognized by different receptors (Ross and Medof, Adv Immunol 37, 217-267 (1985)). This cleavage abolishes the ability of C3b to further amplify C3b deposition and activate the late components of the complement cascade, including the membrane attack complex, capable of direct membrane damage. However, macrophage phagocytic receptors recognize C3b and its fragments preferentially; due to the versatility of the ester-bond formation, C3-mediated opsonization is central to pathogen recognition (Holers et al., Immunol Today 13, 231-236 (1992)), and receptors for the various C3 degradation products therefore play an important role in the host immune response.
C3 itself is a complex and flexible protein consisting of 13 distinct domains. The core of the molecule is made up of 8 so-called macroglobulin (MG) domains, which constitute the tightly packed α and β chains of C3. Inserted into this structure are CUB (C1r/C1s, Uegf and Bone mophogenetic protein-1) and TED domains, the latter containing the thioester bond that allows covalent association of C3b with pathogen surfaces. The remaining domains contain C3a or act as linkers and spacers of the core domains. Comparison of C3b and C3c structures to C3 demonstrate that the molecule undergoes major conformational rearrangements with each proteolysis, which exposes not only the TED, but additional new surfaces of the molecule that can interact with cellular receptors (Janssen and Gros, Mol Immunol 44, 3-10 (2007)).
Complement C3 Receptors on Phagocytic Cells
There are three known gene superfamilies of complement receptors: The short consensus repeat (SCR) modules that code for CR1 and CR2, the beta-2 integrin family members CR3 and CR4, and the immunoglobulin Ig-superfamily member CRIg.
CR1 is a 180-210 kDa glycoprotein consisting of 30 Short Consensus Repeats (SCRs) and plays a major role in immune complex clearance. SCRs are modular structures of about 60 amino acids, each with two pairs of disulfide bonds providing structural rigidity. High affinity binding to both C3b and C4b occurs through two distinct sites, each composed of 3 SCRs) reviewed by (Krych-Goldberg and Atkinson, Immunol Rev 180, 112-122 (2001)). The structure of the C3b binding site, contained within SCR 15-17 of CR1 (site 2), has been determined by MRI (Smith et al., Cell 108, 769-780 (2002)), revealing that the three modules are in an extended head-to-tail arrangement with flexibility at the 16-17 junction. Structure-guided mutagenesis identified a positively charged surface region on module 15 that is critical for C4b binding. This patch, together with basic side chains of module 16 exposed on the same face of CR1, is required for C3b binding. The main function of CR1, first described as an immune adherence receptor (Rothman et al., J Immunol 115, 1312-1315 (1975)), is to capture ICs on erythrocytes for transport and clearance by the liver (Taylor et al., Clin Immunol Immunopathol 82, 49-59 (1997)). There is a role in phagocytosis for CR1 on neutrophils, but not in tissue macrophages (Sengelov et al., J Immunol 153, 804-810 (1994)). In addition to its role in clearance of immune complexes, CR1 is a potent inhibitor of both classical and alternative pathway activation through its interaction with the respective convertases (Krych-Goldberg and Atkinson, 2001, supra; Krych-Goldberg et al., J Biol Chem 274, 31160-31168 (1999)). In the mouse, CR1 and CR2 are two products of the same gene formed by alternative splicing and are primarily associated with B-lymphocytes and follicular dendritic cells and function mainly in regulating B-cell responses (Molina et al., 1996). The mouse functional equivalent of CR1, Crry, inactivates the classical and alternative pathway enzymes and acts as an intrinsic regulator of complement activation rather than as a phagocytic receptor (Molina et al., Proc Natl Acad Sci USA 93, 3357-3361 (1992)).
CR2 (CD21) binds iC3b and C3dg and is the principal complement receptor that enhances B cell immunity (Carroll, Nat Immunol 5, 981-986 (2004); Weis et al., Proc Natl Acad Sci USA 81, 881-885 (1984)). Uptake of C3d-coated antigen by cognate B cells results in an enhanced signal via the B cell antigen receptor. Thus, coengagement of the CD21-CD19-CD81 coreceptor with B cell antigen receptor lowers the threshold of B cell activation and provides an important survival signal (Matsumoto et al., J Exp Med 173, 55-64 (1991)). The CR2 binding site on iC3b has been mapped partly on the interface between the TED and the MG1 domains (Clemenza and Isenman, J Immunol 165, 3839-3848 (2000)).
CR3 and CR4 are transmembrane heterodimers composed of an alpha subunit (CD11b or αM and CD11c or αx, respectively) and a common beta chain (CD18 or β2), and are involved in adhesion to extracellular matrix and to other cells as well as in recognition of iC3b. They belong to the integrin family and perform functions not only in phagocytosis, but also in leukocyte trafficking and migration, synapse formation and costimulation (reviewed by (Ross, Adv Immunol 37, 217-267 (2000)). Integrin adhesiveness is regulated through a process called inside-out signaling, transforming the integrins from a low- to a high-affinity binding state (Liddington and Ginsberg, J Cell Biol 158, 833-839 (2002)). In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm. The binding sites of iC3b have been mapped to several domains on the alpha chain of CR3 and CR4 (Diamond et al., J Cell Biol 120, 1031-1043 (1993); Li and Zhang, J Biol Chem 278, 34395-34402 (2003); Xiong and Zhang, J Biol Chem 278, 34395-34402 (2001)). The multiple ligands for CR3: iC3b, beta-glucan and ICAM-1, seem to bind to partially overlapping sites contained within the I domain of CD11b (Balsam et al., 1998; Diamond et al., 1990; Zhang and Plow, 1996). Its specific recognition of the proteolytically inactivated form of C3b, iC3b, is predicted based on structural studies that locate the CR3 binding sites to residues that become exposed upon unfolding of the CUB domain in C3b (Nishida et al., Proc Natl Acad Sci USA 103, 19737-19742 (2006)), which occurs upon α′ chain cleavage by the complement regulatory protease, Factor I.
CRIg is a macrophage associated receptor with homology to A33 antigen and JAM1 that is required for the clearance of pathogens from the blood stream. A human CRIg protein was first cloned from a human fetal cDNA library using degenerate primers recognizing conserved Ig domains of human JAM1. Sequencing of several clones revealed an open reading frame of 400 amino acids. Blast searches confirmed similarity to Z39Ig, a type 1 transmembrane protein (Langnaese et al., Biochim Biophys Acta 1492 (2000) 522-525). The extracellular region of this molecule was found to consist of two Ig-like domains, comprising an N-terminal V-set domain and a C-terminal C2-set domain. The novel human protein was originally designated as a “single transmembrane Ig superfamily member macrophage associated” (huSTIgMA). (huSTIgMA). Subsequently, using 3′ and 5′ primers, a splice variant of huSTIgMA was cloned, which lacks the membrane proximal IgC domain and is 50 amino acids shorter. Accordingly, the shorter splice variant of this human protein was designated huSTIgMAshort. The amino acid sequence of huSTIgMA (referred to as PRO362) and the encoding polynucleotide sequence are disclosed in U.S. Pat. No. 6,410,708, issued Jun. 25, 2002. In addition, both huSTIgMA and huSTIgMAshort, along with the murine STIgMA (muSTIgMA) protein and nucleic acid sequences, are disclosed in PCT Publication WO 2004031105, published Apr. 15, 2004.
The crystal structure of CRIg and a C3b:CRIg complex is disclosed in U.S. Application Publication No. 2008/0045697, published Feb. 21, 2008.
The Kupffer cells (KCs), residing within the lumen of the liver sinusoids, form the largest population of macrophages in the body. Although KCs have markers in common with other tissue resident macrophages, they perform specialized functions geared towards efficient clearance of gut-derived bacteria, microbial debris, bacterial endotoxins, immune complexes and dead cells present in portal vein blood draining from the microvascular system of the digestive tract (Bilzer et al., Liver Int 26, 1175-1186 (2006)). Efficient binding of pathogens to the KC surface is a crucial step in the first-line immune defense against pathogens (Benacerraf et al., J Exp Med 110, 27-48 (1959)). A central role for KCs in the rapid clearance of pathogens from the circulation is illustrated by the significantly increased mortality in mice depleted of KCs (Hirakata et al., Infect Immun 59, 289-294 (1991)). The identification of CRIg further stresses the critical role of complement and KCs in the first line immune defense against circulating pathogens.
The only complement C3 receptors identified on mouse KCs are CRIg and CR3 (Helmy et al., Cell 124, 915-927 (2006)), while human KCs show additional expression of CR1 and CR4 (Hinglais et al., 1989). Both CRIg and CR3 on KCs contribute to binding to iC3b opsonized particles in vitro (Helmy et al., Lab Invest 61, 509-514 (2006)). In vivo, a role of KC-expressed CR3 in the binding to iC3b-coated pathogens is less clear. CR3 has been proposed to contribute to clearance of pathogens indirectly via recruitment of neutrophils and interaction with neutrophil-expressed ICAMI (Conlan and North, Exp Med 179, 259-268 (1994); Ebe et al., Pathol Int 49, 519-532 (1999); Gregory et al., J Immunol 157, 2514-2520 (1996); Gregory and Wing, J Leukoc Biol 72, 239-248 (2002); Rogers and Unanue, Infect Immun 61, 5090-5096 (1993)). In contrast, CRIg performs a direct role by capturing pathogens that transit through the liver sinusoidal lumen (Helmy et al., 2006, supra). A difference in the biology of CRIg vs CR3 is in part reflected by difference in binding characteristics of these two receptors. CRIg expressed on KCs constitutively binds to monomeric C3 fragments whereas CR3 only binds to iC3b-opsonized particles (Helmy et al., 2006, supra). The capacity of CRIg to efficiently capture monomeric C3b and iC3b as well as C3b/iC3b-coated particles reflects the increased avidity created by a multivalent interaction between CRIg molecules concentrated at the tip of membrane extensions of macrophages (Helmy et al., 2006, supra) and multimers of C3b and iC3b present on the pathogen surface. While CR3 only binds iC3b-coated particles, CRIg additionally bind to C3b, the first C3 cleavage product formed on serum-opsonized pathogens (Croize et al., Infect Immun 61, 5134-5139 (1993)). Since a large number of C3b molecules bound to the pathogen surface are protected from cleavage by factor H and I (Gordon et al., J Infect Dis 157, 697-704 (1988)), recognition of C3b ligands by CRIg ensures rapid binding and clearance. Thus, while both CRIg and CR3 are expressed on KCs, they show different ligand specificity, distinct binding properties and distinct kinetics of pathogen clearance.
Examples of pathogens that exploit cell surface receptors for cellular entry are viruses like human immunodeficiency virus (HIV), and intracellular bacteria like Mycobacterium tuberculosum, Mycobacterium leprae, Yersinia pseudotuberculosis, Salmonella typhimurium and Listeria Monocytogenes and parasites like the prostigmatoid Leishmania major (Cossart and Sansonetti, Science 304:242-248 (2004); Galan, Cell 103:363-366 (2000); Hornef et al., Nat. Immunol. 3:1033-1040 (2002); Stoiber et al., Mol. Immunol. 42:153-160 (2005)).
As discussed above, CRIg is a recently discovered complement C3 receptor expressed on a subpopulation of tissue resident macrophages. Next to functioning as a complement receptor for C3 proteins, the extracellular IgV domain of CRIg selectively inhibits the alternative pathway of complement by binding to C3b and inhibiting proteolytic activation of C3 and C5. However, CRIg binding affinity for the convertase subunit C3b is low (IC50>1 μM) requiring a relatively high concentration of protein to reach near complete complement inhibition. Accordingly, there is a need for CRIg polypeptides with improved therapeutic efficacy. The present invention provides such polypeptides.